NPP1 fusion proteins

ABSTRACT

The present invention provides a novel fusion polypeptide containing a catalytic portion of NPP1 fused to a targeting moiety, nucleic acids encoding the fusion polypeptide, a vector containing the nucleic acid integrated thereinto, a host cell transformed with the vector and pharmaceutical compositions comprising the fusion polypeptide.

RELATED APPLICATION

This application claims the benefit of International Publication No.PCT/US2011/028233, filed on Mar. 11, 2011, and U.S. ProvisionalApplication No. 61/340,066, filed on Mar. 12, 2010. The entire teachingsof each of the above applications are expressly incorporated herein byreference.

Submitted herewith is a text file corresponding to the amino acid andnucleotide sequence numbers 1-18 (SEQ ID NOs: 1-18) entitled“Sequence_Listing_ST252_1.TXT”. The file was created on Jun. 6, 2013 andis 84,371 bytes in size. The material in this file is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/ENPP1/PC-1) isa type II transmembrane glycoprotein that forms a homodimer. The proteincleaves a variety of substrates, including phosphodiester bonds ofnucleotides and nucleotide sugars and pyrophosphate bonds of nucleotidesand nucleotide sugars. NPP1 protein functions to hydrolyze nucleoside 5′triphosphtase to either corresponding monophosphates and also hydrolyzesdiadenosine polyphosphates. Mutations in the NPP1 gene have beenassociated with idiopathic infantile arterial calcification (IIAC),insulin resistance, hypophosphatemic rickets, and ossification of theposterior longitudinal ligament of the spine.

IIAC, a rare autosomal recessive and nearly always fatal disorder, ischaracterized by calcification of the internal elastic lamina ofmuscular arteries and stenosis due to myointimal proliferation. Thereare more than 160 cases of IIAC that have been reported world-wide. Thesymptoms of the disease most often appear by early infancy, and thedisease is lethal by 6 months of age, generally because of ischemiccardiomyopathy, and other complications of obstructive arteriopathyincluding renal artery stenosis. In more than a dozen reported cases ofIIAC, periarticular calcifications of large joints also developed ininfancy. Rutsch et al. (2003) reported that mutations in ENPP1 areassociated with IIAC characterized by the spontaneous periarticular andaortic calcifications in early life and systemic lowering of nucleotidepyrophosphatase/phosphodiesterase activity in the affected individuals.

Although defects in the NPP1 protein have been implicated in suchserious disease as IIAC, there is no treatment available in the art forthose who are affected by the disease. Therefore, a dire need exists foran effective and safe composition, formulation and medicament for thetreatment of IIAC, insulin resistance, hypophosphatemic rickets, andossification of the posterior longitudinal ligament of the spine.

SUMMARY OF THE INVENTION

The present invention includes fusion proteins of truncated domains ofNPP1 (i.e., an NPP1 component) fused to a targeting moiety. Thetargeting moiety functions to enhance the efficiency in targeting theNPP1 fusion protein to a site clinical or biological importance (e.g.,site of calcification in a subject that needs lowering ofcalcification). Without wishing to limit the invention to any particulartheory or mechanism of operation it is believed that the NPP1 componentfunction to inhibit calcification by enhancing the formation ofpyrophosphate (PPi) and/or by cleaving pyrophosphate to produce solublephosphate (Pi) and/or by increasing the availability of adenosinemonophosphate (AMP) and/or adenosine. It is contemplated that thetargeting moiety can be attached to the N-terminus and/or the C-terminusof the NPP1 component at any useful position. Additionally, the NPP1fusion protein disclosed herein can also include one or more of Fcfragment, PEG, polypeptide linker or other additional polypeptides toenhance the enzymatic activity, stability or targeting.

The fusion proteins of the invention can be used to treat a wide varietyof conditions in a subject. Any condition that can be beneficiallytreated by the administering of a fusion protein of the invention isincluded within the scope of the invention. For example, treatment ofconditions that can be improved by reducing and/or eliminating one ormore calcification structures and/or preventing calcification structuresfrom forming in a subject such as a mammal, for example, a human patientis within the scope of the invention. Conditions such as arterialblockage are contemplated for treatment by employing fusion proteins ofthe invention. In one particularly useful embodiment, the condition tobe treated is generalized arterial calcification of infancy alsoreferred to as idiopathic arterial calcification of infancy and arterialmedia calcification of infancy. Conditions such as insulin resistance,hypophosphatemic rickets, and ossification of the posterior longitudinalligament of the spine are also contemplated for treatment.

Fusion proteins of the invention can be produced in any useful proteinexpression system including, without limitation, cell culture (e.g., CHOcells, COS cells, HEK203), bacteria such as Escherichia coli (E. coli)and transgenic animals, including, but no limited to, mammals and avians(e.g., chickens, quail, duck and turkey).

The manufacture of pharmaceutical compositions (or pharmaceuticalformulations) is well known in the art and such pharmaceuticalcompositions are contemplated for use in accordance with fusion proteinsof the invention.

Generally, the dosage of fusion protein administered to a subject willvary depending upon known factors such as age, health and weight of therecipient, type of concurrent treatment, frequency of treatment, and thelike. Usually, a dosage of active ingredient (i.e., fusion protein) canbe between about 0.0001 and about 50 milligrams per kilogram of bodyweight. Precise dosage, frequency of administration and time span oftreatment can be determined by a physician skilled in the art ofadministration of therapeutic proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of wild-type NPP1 protein.The cytosolic and transmembrane regions are underlined. The potentialN-glycosylation sites are in bold. PSCAKE in italics is the start ofsoluble NPP1 which includes the cysteine rich region.

FIG. 2 illustrates the amino acid sequence of the catalytic domain(s) ofNPP1 protein having no target moiety attached (“sssNPP1”).

FIG. 3 illustrates the amino acid sequence of TAGsssNPP1.The targetingmoiety of eight consecutive aspartic acid residues is fused to theN-terminus of the sssNPP1.The signal peptide is underlined and thetargeting moiety is indicated in bold.

FIG. 4 illustrates the amino acid sequence of TAGsssNPP1 that containsthe targeting moiety of ten consecutive aspartic acid residues fused tothe C-terminus of the sssNPP1.The signal peptide is underlined and thetargeting moiety is indicated in bold.

FIG. 5 illustrates the nucleic acid sequence of TAGssNPP1 fusionprotein. The targeting moiety of eight consecutive aspartic acidresidues is fused to the N-terminus of the ssNPP1.

FIG. 6 illustrates the amino acid sequence of TAGssNPP1 fusion protein.The targeting moiety of eight consecutive aspartic acid residues isfused to the N-terminus of the ssNPP1.The signal peptide is underlinedand the targeting moiety is indicated in bold.

FIG. 7 illustrates the nucleic acid sequence of ssNPP1.

FIG. 8 illustrates the amino acid sequence of ssNPP1.The signal peptideis underlined.

FIG. 9 illustrates the nucleic acid sequence of sNPP1.

FIG. 10 illustrates the amino acid sequence of sNPP1.The signal peptideis underlined.

FIG. 11 illustrates the nucleic acid sequence of TAGsNPP1.The targetingmoiety of eight consecutive aspartic acid residues is fused to theN-terminus of the sNPP1.

FIG. 12 illustrates the amino acid sequence of TAGsNPP1.The targetingmoiety of eight consecutive aspartic acid residues is fused to theN-terminus of the ssNPP1.The signal peptide is underlined and thetargeting moiety is indicated in bold.

FIG. 13 illustrates the nucleic acid sequence of TAGsNPP1.The targetingmoiety of eight consecutive aspartic acid residues is fused to theC-terminus of the sNPP1.

FIG. 14 illustrates the amino acid sequence of TAGsNPP1.The targetingmoiety of eight consecutive aspartic acid residues is fused to theN-terminus of the sNPP1.The signal peptide is underlined and thetargeting moiety is indicated in bold.

FIG. 15 illustrates the amino acid sequence of a linker peptide.

FIG. 16 illustrates the amino acid sequence of an immunoglobulin Fcsegment.

FIG. 17 illustrates the amino acid sequence of TAGsssNPP1 which containsthe targeting moiety of eight consecutive aspartic acid residues fusedto the N-terminus of the sssNPP1 via a peptide linker. The Fc segment isfused to N-terminus of the target moiety. The signal peptide isunderlined and the targeting moiety is indicated in bold. The peptidelinker is in italics.

FIG. 18 illustrates the amino acid sequence of TAGsssNPP1 which containsthe targeting moiety of eight consecutive aspartic acid residues fusedto the C-terminus of the sssNPP1 via a peptide linker. The Fc segment isfused to N-terminus of the sssNPP1.The signal peptide is underlined andthe targeting moiety is indicated in bold. The peptide linker is initalics.

FIG. 19 is a schematic representation of an expression vector (i.e.,pTT22) containing a TAGsNPP1 construct. The targeting moiety of eightconsecutive aspartic acid residues fused to the C-terminus of the sNPP1.

FIG. 20 illustrates Western blot analysis of TAGsNPP1.R, reducingcondition; NR, non-reducing condition.

FIG. 21 demonstrates the enzymatic activity of TAGsNPP1 produced andisolated from HEK293 cells.

FIGS. 22A-22C illustrate schematics of TAGNPP1 fusion protein constructsdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel human NPP1 fusion proteins that aresoluble and contain truncated and biologically active domain(s) of NPP1(i.e., NPP1 components that contain at least one extracellular catalyticdomain of naturally occurring NPP1 for the pyrophosphatase and/orphosphodiesterase activity) and one or more targeting moieties (i.e.,“TAG”). The NPP1 fusion proteins of the present invention comprise atleast the NPP1 domain essential to carry out the pyrophosphatase and/orphosphodiesterase activity. Accordingly, the invention features isolatedfusion proteins comprising the amino acid residues A205 through L591 ofSEQ ID NO:1 fused to one or more targeting moieties. The targetingmoiety can be recombinantly fused or chemically bonded (e.g., covalentbond, ionic bond, hydrophobic bond and Van der Waals force) to the NPP1component by methods well known in the art and direct the NPP1 componentto a certain target site where the attached NPP1 component will have adesirable effect (e.g., catalysis of a reaction such as solubilizing asubstrate such as PPi or preventing the formation of a substrate such asPPi) in a subject to which the fusion protein of the present inventionis administered.

TAGNPP1s

All NPP1 fusion proteins (“TAGNPP1s”) of the present invention have theN-terminal cytosolic and the transmembrane domains of the naturallyoccurring human NPP1 removed. Optionally, TAGNPP1s fusion proteins ofthe present invention can also contain C-terminal truncation ofwild-type NPP1 in various lengths. The amino acid sequence offull-length wild-type NPP1 is set forth in SEQ ID NO: 1.

In one embodiment, the fusion protein contains a polypeptide comprisingthe amino acid residues A205 through L591 of SEQ ID NO:1 (“sssNPP1”)fused a TAG on either the N- or C-terminus of the polypeptide(“TAGsssNPP1”). In one embodiment, the fusion protein comprises apolypeptide comprising the amino acid residues A205 through D925 of SEQID NO:1 (“ssNPP1”) fused to a TAG on either the N- or C-terminus(“TAGssNPP1”). In one embodiment, the fusion protein comprises apolypeptide comprising the amino acid residues P99 through D925 of SEQID NO:1 (“sNPP1”) fused to a TAG on either the N- or C-terminus of thepolypeptide (“TAGsNPP1”). Also contemplated is any consecutive fragmentof sNPP1 that comprises at least the amino acid resides A205 throughL591 of SEQ ID NO:1 and the polypeptide fragment is fused to a TAG oneither the N- or C-terminus.

When expressed in a cell culture or transgenic animal, the TAGNPP1fusion proteins can further comprise a signal peptide (or leadersequence) at its N-terminus. The signal peptide co-translationally orpost-translationally directs transport of the TAGNPP1 fusion proteinsthrough the subcellular organelles of the cell expressing the TAGNPP1fusion proteins and, thereby determining the post-translationalmodification of the TAGNPP1 fusion proteins. It is to be understood thatbecause the signal peptide is cleaved at the co-translational orpost-translational stage of the fusion protein, the TAGNNP1 fusionproteins are generally devoid of the signal peptide when once secretedand isolated. Accordingly, in the embodiments that are directed to thenucleic acid sequences encoding the TAGNPP1 fusion proteins aredescribed, the leader sequences are also contemplated as used in thepresent invention. For example, the nucleotide sequence set forth in SEQID NO:2 contains an example of the leader sequence for TAGNNP1 at its 5′end.

Each of the fusion proteins disclosed herein is contemplated with one ormore targeting moieties (“TAG”). The TAG component according to thepresent invention comprises four or more negatively charged amino acidssuch as aspartic acid and glutamic acid. The TAG component can be astretch of negatively charged amino acid residues, for example, asparticacids and/or glutamic acids that are between about 4 and about 20 aminoacid residues in length. The TAG can be fused to either the N- orC-terminus of the NPP1 component. The TAG can be also fused to both N-and C-termini of the NPP1 component. Accordingly, the amino acidsequence of the TAGsNPP1 fusion protein, for example, includes PSCAKEthrough the C-terminus end of the NPP1 component and one or moretargeting moieties (e.g., polyglutamic acid tag or polyaspartic acidtag) at the N- and/or C-terminus end of the NPP1 component. The fusionprotein comprising the NPP1 component having the TAG fused to theC-terminus is a particularly useful embodiment. In one very specificembodiment, TAG having a stretch of eight aspartic acids is employed ascan be seen in the exemplary sequences for TAGsNPP1 and TAGssNPP1 inFigures, though any useful number of negatively charged amino acidresidues (e.g., 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17,or 18) canbe used in accordance with the invention. The TAG component is indicatedas “A” in Figures.

The invention also encompasses polynucleotides which encode variousTAGNPP1 fusion proteins described herein. Accordingly, any nucleic acidsequence which encodes the amino acid sequence of any TAGNPP1 fusionprotein can be used to generate recombinant molecules which express thecorresponding TAGNPP1 fusion protein. In a particular embodiment, theinvention encompasses the polynucleotide comprising the nucleic acidsequence of SEQ ID NO:2 as shown in FIG. 2.

Within certain specific embodiments, the fusion protein comprisesmultiple polypeptides as described herein, or that comprises at leastone polypeptide as described herein and an unrelated sequence. Certainpreferred polypeptide can, for example, assist in dimerization andstability or minimize aggregation of the fusion proteins. For example,the additional polypeptide can be the Fc region of the immunoglobulin G1to increase stability in serum. The use of the Fc segment is well knownin the art and described in U.S. Pat. Nos. 7,902,151; and 7,858,297,theentire teachings of which are incorporated herein by reference in theirentirety. The cysteine rich region of wild-type NPP1 (i.e., PSCAKEthrough NEPQCP; the amino acid sequence from P99 to P204 of SEQ ID NO:1)can be employed to facilitates dimerization of the TAGNPP1 fusionproteins.

In another embodiment, the polyethylene glycol (PEG) can be conjugatedto the TAGNPP1 fusion proteins. Other polypeptides may be selected so asto minimize aggregation and immunogenicity, to increase the solubilityof the protein, or to enable the protein to be targeted to desired sitesof clinical or biological importance.

TAGNPP1 can be also fused or conjugated to an appropriate polypeptidelinker or other sequence for ease of identification, synthesis, orpurification of the fusion protein, or to better preserve the nativestructure of the NPP1 component which can enhance the activity andtargeting of the TAGNPP1.Specifically, a peptide linker sequence can beemployed to separate the first and the second polypeptide components bya distance sufficient to ensure that each polypeptide folds into itssecondary and tertiary structures. Such a peptide linker sequence isincorporated into the fusion protein between the NPP1 component and theTAG component using standard techniques well known in the art. Suitablepeptide linker sequences may be chosen based on their ability to adopt aflexible extended conformation and their inability to adopt a secondarystructure that could interact with functional portion on the NPP1,TAG orother secondary polypeptides described herein (e.g., Fc). Preferredpeptide linker sequences contain Gly, His, Asn and Ser residues. Theuseful peptide linkers include, without limitation, poly-Gly, poly-His,poly-Asn, or poly-Ser. Other near neutral amino acids, such as Thr andAla can be also used in the linker sequence. Amino acid sequences whichcan be usefully employed as linkers include those disclosed in Marateaet al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad Sci.USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180.The linkersequence may be from 1 to about 20 amino acid residues in length.Preferably, the polypeptide linker is between about 8 and about 12 aminoacids in length. In a preferred embodiment, the peptide linker used inthe invention is GGGGSGGGGS (SEQ ID NO:15), although any functionalcombination of Gly, Ser, His, or Asn can be employed.

Fusion proteins can also comprise a TAGNPP1 of the present inventiontogether with an unrelated polypeptide. Preferably the unrelatedpolypeptide is capable of enhancing the targeting of the fusion proteinto the site of clinical or biological importance (e.g., site ofcalcification). For example, peptides that have high affinity to thebone are described in U.S. Pat. No. 7,323,542,the entire teachings ofwhich are incorporated herein by reference.

TAGNPP1 can be prepared using standard methods, including recombinanttechniques or chemical conjugation well known in the art. Techniquesuseful for isolating and characterizing the nucleic acids and proteinsof the present invention are well known to those of skill in the art andstandard molecular biology and biochemical manuals can be consulted toselect suitable protocols for use without undue experimentation. See,for example, Sambrook et al, 1989,“Molecular Cloning: A LaboratoryManual,” 2nd ed., Cold Spring Harbor, the content of which is hereinincorporated by reference in its entirety. Briefly, DNA sequencesencoding the polypeptide components can be assembled separately, andligated into an appropriate expression vector. For example, the 3′ endof the DNA sequence encoding the NPP1 component is ligated, with orwithout a peptide linker, to the 5′ end of a DNA sequence encoding thesecond polypeptide component such as TAG PEG, or Fc so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides. The ligated DNA sequences are operably linked tosuitable transcriptional or translational regulatory elements includinga promoter. The regulatory elements responsible for expression of DNAare located only 5′ to the DNA sequence encoding the first polypeptidesuch as the leader sequence encoding a signal peptide. Similarly, stopcodons required to end translation and transcription termination signalsare only present 3′ to the DNA sequence encoding the second polypeptide.

The invention also encompasses TAGNPP1 variants. A preferred TAGNPP1variant is one having 80%, 85%, 90%, 95% and more preferably 96% aminoacid sequence identity to the amino acid sequence A205 through L591 ofSEQ ID NO:1. A most preferred TAGNPP1 variant is one having at least 97%amino acid sequence identity to amino acid sequence A205 through L591 ofSEQ ID NO:1.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, the presentinvention also features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2 and whose the antisense sequence is85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:2.Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex or probe, as taught in Wahl, G. M. and S. L. Berger(1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; MethodsEnzymol. 152:507-511), and can be used at a defined stringency.

The invention additionally contemplates nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof.

Although nucleotide sequences which encode TAGNPP1 and its variants arepreferably capable of hybridizing to the nucleotide sequence of theTAGNPP1 under appropriately selected conditions of stringency, it can beadvantageous to produce nucleotide sequences encoding TAGNPP1 or itsderivatives possessing a substantially different codon usage. Codons canbe selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding TAGNPP1 and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life.

Altered nucleic acid sequences encoding TAGNPP1 which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent of TAGNPP1.The encoded protein canalso contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent TAGNPP1.Deliberate amino acid substitutions can be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of TAGNPP1 is retained. For example, positivelycharged amino acid residues include Lys and Arg; negatively chargedamino acid residues include Asp and Glu; and amino acids with unchargedpolar head groups having similar hydrophilicity can include Leu, Ile,and Val; Gly and Ala; Asp and Gln; Ser and Thr; Phe and Tyr.

Expression Vector

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing sequences encoding TAGNPP1 andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook, J. et al. (1989) Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., the teachings of which areincorporated herein by reference in its entirety.

A variety of expression vector/host systems can be utilized to containand express sequences encoding TAGNPP1.These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems(e.g., pTT22 vector).

The control elements or regulatory sequences can includes thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions-which interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including, tissue-specific, constitutive and inducible promoters, can beused. For example, when cloning in bacterial systems, induciblepromoters such as the hybrid lacZ promoter of the Bluescript™ phagemid(Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and thelike can be used. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferred. If it is necessary togenerate a cell line that contains multiple copies of the sequenceencoding TAGNPP1,vectors based on SV40 or EBV can be used with anappropriate selectable marker. When an avian expression system is used,suitable vectors for expression various TAGNPP1 constructs are describedin U.S. Pat. Nos. 6,730,822; 6,825,396; 6,875,588; 7,294,507; 7,521,591;7,534,929; and U.S. patent application Ser. No. 11/376,023,the entireteachings of which are incorporated herein by reference in theirentirety. Briefly, when an avian expression system is employed toexpress TAGNPP1,suitable oviduct-specific promoters, for example, andwithout limitation, ovomucoid promoters, ovalbumin promoters, lysozymepromoters, conalbumin promoters, ovomucin promoters, ovotransferrinpromoters and functional portions of each of these promoters arecontemplated. Suitable non-specific promoters can include, for exampleand without limitation, cytomegalovirus (CMV) promoters, MDOT promotersand rous-sarcoma virus (RSV) promoters, murine leukemia virus (MLV)promoters, mouse mammary tumor virus (MMTV) promoters and SV40 promotersand functional portions of each of these promoters. Non-limitingexamples of other promoters which can be useful in the present inventioninclude, without limitation, Pol III promoters (for example, type 1,type2 and type 3 Pol III promoters) such as H1 promoters, U6 promoters, tRNApromoters, RNase MPR promoters and functional portions of each of thesepromoters. Typically, functional terminator sequences are selected foruse in the present invention in accordance with the promoter that isemployed.

Host Cells

The present invention includes the production of soluble TAGNPP1 in atransgenic avian (e.g., transgenic chicken) system as is well known inthe art, for example, in U.S. Pat. No. 7,534,929,the disclosure of whichis incorporated in its entirety herein by reference. Production in theavian system (e.g., in the avian oviduct) of an NPP1 component with orwithout to a targeting moiety (e.g., ssNPP1, sNPP1,TAGsNPP1 andTAGssNPP1) is within the scope of the invention. Furthermore, TAGNPP1produced in any useful protein expression system including, withoutlimitation, transgenic avians, transgenic mammal, cell culture (e.g.,CHO cells, HEK293 cells, and COS cells), bacteria such as E.coli,transgenic animals such as mammals and avians (e.g., chickens,quail, duck and turkey) and in plant systems including duck weed, iscontemplated herein.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedTAGNPP1s in the desired fashion. Such modifications of the polypeptideof TAGNNP1 include, without limitation, acetylation, carboxylation,sialylation, glycosylation, phosphorylation, lipidation, and acylation.Different host cells such as CHO, COS, HeLa, MDCK, HEK293,and W138,whichhave specific cellular machinery and characteristic mechanisms for suchpost-translational activities, can be chosen to ensure the correctmodification and processing of the fusion protein of the presentinvention. Avian tumor cell line is also contemplated as a host cell forexpressing the polypeptide of the present invention. Examples of usefulavian cell lines (e.g., an avian oviduct tumor cell line) which can beemployed in the present invention are described in U.S. Pat. PublicationNo. 2009/0253176,the entire teachings of which are incorporated hereinby reference.

Production of TAGNPP1

TAGNPP1 can be produced using any of a variety of well-known techniques.TAGNPP1 encoded by DNA sequences as described above can be readilyprepared from the DNA sequences using any of a variety of expressionvectors described herein or well known to those of ordinary skill in theart. Expression can be achieved in any appropriate host cell that hasbeen transformed or transfected with an expression vector containing aDNA molecule that encodes a recombinant polypeptide of the presentinvention. Supernatants from suitable host/vector systems which secreterecombinant fusion protein or polypeptide into culture media can befirst concentrated using a commercially available filter. Followingconcentration, the concentrate can be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. One or morereverse phase HPLC steps can be employed to further purify a recombinantpolypeptide.

For high-yield production of recombinant proteins, stable expression ispreferred. Cell lines stably expressing TAGNPP1 can be transformed usingexpression vectors which contain viral origins of replication and/orendogenous expression elements and/or a selectable marker gene on thesame or on a separate vector. Following the introduction of the vector,cells can be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltype. Methods of producing exogenous protein in mammalian cell lines arewell known in the art. Illustrative examples of this and other aspectsand embodiments of the present invention for the production ofheterologous polypeptides such as TAGNPP1 fusion proteins in avian cellsare fully disclosed in U.S. patent application Ser. No. 09/877,374,filedJun. 8, 2001,published as U.S. 2002/0108132-A1 on Aug. 8, 2002,and U.S.patent application Ser. No. 10/251,364,filed Sep. 18, 2002,each of whichare incorporated herein by reference in their entirety. Examples ofproducing exogenous proteins in avian tumor cell lines are alsodescribed in U.S. Pat. Publication No. 2009/0253176,the entire teachingsof which are incorporated herein by reference in entirety.

The invention specifically contemplates the production of the TAGNPP1proteins disclosed herein in a transgenic avian system. In oneparticularly useful embodiment, the invention is drawn to the productionof TAGNPP1 which can be produced in the oviduct of a transgenic avian,such as a chicken, in accordance with the invention. Examples ofproducing exogenous proteins in transgenic avian expression system arealso described in U.S. Pat. No. 6,730,822,the entire teachings of whichare incorporated herein by reference in entirety. Briefly, a suitableavian vector described above that contains a nucleic acid sequenceencoding a TAGNPP1 fusion protein, operably linked to a tissue-specificor constitutive promoter that drives expression of the encoding sequencein the chicken oviduct are introduced into chicken stage X embryoniccells. The transformed embryonic cells are incubated under conditionsconducive to hatching live chicks. Live chicks are nurtured into amature chimeric chicken which are mated with a non-transgenic chickennaturally or via artificial insemination. A transgenic chicken isidentified by screening progeny for germ line incorporation of theprotein encoding sequence. The transgenic progeny can be mated withanother transgenic or a non-transgenic chicken to produce eggscontaining the TAGNPP1 fusion protein. The TAGNPP1 is then isolated andpurified by methods well known in the art. Accordingly, the inventionprovides recombinant TAGNPP1 fusion proteins that have been produced bytransgenic avians.

Pharmaceutical Composition

The present invention also features pharmaceutical compositionscomprising isolated and substantially purified TAGNPP1 or apharmaceutically acceptatable salt thereof. Pharmaceutical compositionof the present invention can also include a pharmaceutically acceptablecarrier or expient therefor. Compositions comprising such carriers,including composite molecules, are formulated by well-known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 14thEd., Mack Publishing Co., Easton, Pa.), the entire teachings of whichare incorporated herein by reference. The carrier may comprise adiluent. In one embodiment, the pharmaceutical carrier can be a liquidand the fusion protein may be in the form of a solution. Thepharmaceutical carrier can be wax, fat, or alcohol. In anotherembodiment, the pharmaceutically acceptable carrier may be a solid inthe form of a powder, a lyophilized powder, or a tablet. In oneembodiment, the carrier may comprise a liposome or a microcapsule.

The pharmaceutical compositions can be in the form of a sterilelyophilized powder for injection upon reconstitution with a diluent. Thediluent can be water for injection, bacteriostatic water for injection,or sterile saline. The lyophilized powder may be produced by freezedrying a solution of the fusion protein to produce the protein in dryform. As is known in the art, the lyophilized protein generally hasincreased stability and a longer shelf life than a liquid solution ofthe protein.

Definitions:

As used herein, the term “acceptable” with respect to a formulation,composition or ingredient, as used herein, means having no persistentdetrimental effect on the general health of the subject being treated.

As used herein, the term “administration” or “administering” refers toproviding a fusion protein of the invention to a subject in need oftreatment.

“Alterations,” as used herein, comprise any alteration in the sequenceof polynucleotides encoding TAGNPP1 including deletions, insertions, andpoint mutations that may be detected using hybridization assays.

The term “animal” is used herein to include all vertebrate animals,including avians and mammals such as rat, mouse and human. It alsoincludes an individual animal in all stages of development, includingembryonic and fetal stages.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a fusion protein molecule, “amino acid sequence” and liketerms, such as “polypeptide” or “protein” are not meant to limit theamino acid sequence to the complete amino acid sequence associated withthe recited protein or polypeptide molecule.

The term “avian” as used herein refers to any species, subspecies orstrain of organism of the taxonomic class ava, such as, but not limitedto, such organisms as chicken, turkey, duck, goose, quail, pheasants,parrots, finches, hawks, crows and ratites including ostrich, emu andcassowary. The term includes the various known strains of Gallus gallus,or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock,Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox,California Gray, Italian Partridge-colored), as well as strains ofturkeys, pheasants, quails, duck, ostriches and other poultry commonlybred in commercial quantities.

The phrase “based on” or “derived from” as in a retroviral vector beingbased on or derived from a particular retrovirus or based on anucleotide sequence of a particular retrovirus mean that the genome ofthe retroviral vector contains a substantial portion of the nucleotidesequence of the genome of the particular retrovirus. The substantialportion may be a particular gene or nucleotide sequence such as thenucleotide sequence encoding the gag, pol and/or env proteins or otherstructural or functional nucleotide sequence of the virus genome such assequences encoding the LTRs or may be substantially the completeretrovirus genome, for example, most (e.g., more than 60% or more than70% or more than 80% or more than 90%) or all of the retrovirus genome,as will be apparent from the context in the specification as theknowledge of one skilled in the art. Examples of retroviral vectors thatare based on or derived from a retrovirus are the NL retroviral vectors(e.g., NLB) which are based on the ALV retrovirus as disclosed in Cossetet al., Journal of Virology (1991) vol 65,p 3388-3394.

The term “biologically active,” as used herein, refers to a fusionprotein having structural, regulatory, or biochemical functions ofpyrophosphatase/phosphodiesterase of a naturally occurring NPP1 protein.

The term “construct,” as used herein, refers to a linear or circularnucleotide sequence such as DNA that has been assembled from more thanone segments of nucleotide sequence which have been isolated from anatural source or have been chemically synthesized, or combinationsthereof.

The term “complementary,” as used herein, refers to two nucleic acidmolecules that can form specific interactions with one another. In thespecific interactions, an adenine base within one strand of a nucleicacid can form two hydrogen bonds with thymine within a second nucleicacid strand when the two nucleic acid strands are in opposingpolarities. Also in the specific interactions, a guanine base within onestrand of a nucleic acid can form three hydrogen bonds with cytosinewithin a second nucleic acid strand when the two nucleic acid strandsare in opposing polarities. Complementary nucleic acids as referred toherein, may further comprise modified bases wherein a modified adeninemay form hydrogen bonds with a thymine or modified thymine, and amodified cytosine may form hydrogen bonds with a guanine or a modifiedguanine

A “deletion,” as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

The term “expressed” or “expression” as used herein refers to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein canalso refer to the translation of RNA to produce a protein or peptide.

The term “expression vector” as used herein refers to a nucleic acidvector that comprises a gene expression controlling region, such as apromoter or promoter component, operably linked to a nucleotide sequencecoding at least one polypeptide.

“Functional portion” or “functional fragment” are used interchangeablyand as used herein means a portion or fragment of a whole capable ofperforming, in whole or in part, a function of the whole. For example, abiologically functional portion of a molecule means a portion of themolecule that performs a biological function of the whole or intactmolecule. For example, a functional portion of a gene expressioncontrolling region is a fragment or portion of the specified geneexpression controlling region that, in whole or in part, regulates orcontrols gene expression (e.g., facilitates either in whole or in part)in a biological system (e.g., a promoter). Functional portions may be ofany useful size.

The term “gene expression controlling region” as used herein refers tonucleotide sequences that are associated with a coding sequence andwhich regulate, in whole or in part, expression of the coding sequence,for example, regulate, in whole or in part, the transcription of thecoding sequence. Gene expression controlling regions may be isolatedfrom a naturally occurring source or may be chemically synthesized andcan be incorporated into a nucleic acid vector to enable regulatedtranscription in appropriate cells. The “gene expression controllingregions” may precede, but is not limited to preceding, the region of anucleic acid sequence that is in the region 5′ of the end of a codingsequence that may be transcribed into mRNA.

The terms “heterologous,” “exogenous” and “foreign” are usedinterchangeably herein and in general refer to a biomolecule such as anucleic acid or a protein that is not normally found in a certainorganism or in a certain cell, tissue or other component contained in orproduced by an organism. For example, a protein that is heterologous orexogenous to an egg is a protein that is not normally found in the egg.As used herein, the terms “heterologous,” “exogenous” and “foreign” withreference to nucleic acids, such as DNA and RNA, are usedinterchangeably and refer to nucleic acid that does not occur naturallyas part of a chromosome, a genome or cell in which it is present orwhich is found in a location(s) and/or in amounts that differ from thelocation(s) and/or amounts in which it occurs in nature. It can benucleic acid that is not endogenous to the genome, chromosome or celland has been exogenously introduced into the genome, chromosome or cell.Examples of heterologous DNA include, but are not limited to, a DNAcomprising a gene expression control region and DNA that encodes aproduct or products, for example, RNA or protein product. Examples ofheterologous DNA include, but are not limited to, gene expressioncontrolling regions or promoters disclosed herein once isolated from theavian and as used thereafter, e.g., after re-introduction into an aviangenome.

The term “isolated nucleic acid” as used herein covers, for example, (a)a DNA which has the sequence of part of a naturally occurring genomicmolecule but is not flanked by at least one of the sequences that flankthat part of the molecule in the genome of the species in which itnaturally occurs; (b) a nucleic acid which has been incorporated into avector or into the genomic DNA of a prokaryote or eukaryote in a mannersuch that the resulting vector or genomic DNA is not identical tonaturally occurring DNA from which the nucleic acid was obtained; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), ligase chain reaction (LCR)or chemical synthesis, or a restriction fragment; (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein, and (e) a recombinant nucleotide sequence that is partof a hybrid sequence that is not naturally occurring. Isolated nucleicacid molecules of the present invention can include, for example,natural allelic variants as well as nucleic acid molecules modified bynucleotide deletions, insertions, inversions, or substitutions.

An “insertion” or “addition,” as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to theTAGNPP1 molecule.

The term “nucleic acid” as used herein refers to any linear orsequential array of nucleotides and nucleosides, for example cDNA,genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides andderivatives thereof. For ease of discussion, non-naturally occurringnucleic acids may be referred to herein as constructs. Nucleic acids caninclude bacterial plasmid vectors including expression, cloning, cosmidand transformation vectors such as, animal viral vectors such as, butnot limited to, modified adenovirus, influenza virus, polio virus, poxvirus, retroviruses such as avian leukosis virus (ALV) retroviralvector, a murine leukemia virus (MLV) retroviral vector, and alentivirus vector, and the like and fragments thereof. In addition, thenucleic acid can be an LTR of an avian leukosis virus (ALV) retroviralvector, a murine leukemia virus (MLV) retroviral vector, or a lentivirusvector and fragments thereof. Nucleic acids can also include NL vectorssuch as NLB, NLD and NLA and fragments thereof and syntheticoligonucleotides such as chemically synthesized DNA or RNA. Nucleicacids can include modified or derivatised nucleotides and nucleosidessuch as, but not limited to, halogenated nucleotides such as, but notonly, 5-bromouracil, and derivatised nucleotides such as biotin-labelednucleotides.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.

The term “operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Gene expression controlling regions or promoters (e.g.,promoter components) operably linked to a coding sequence are capable ofeffecting the expression of the coding sequence. The controllingsequences need not be contiguous with the coding sequence, so long asthey function to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “oviduct specific promoter” as used herein refers to promotersand promoter components which are functional, i.e., provide fortranscription of a coding sequence, to a large extent, for example,primarily (i.e., more than 50% of the transcription product produced inthe animal by a particular promoter type being produced in oviductcells) or exclusively in oviduct cells of a bird. Examples of usefuloviduct specific promoters include, without limitation, ovalbuminpromoter, ovomucoid promoter, ovoinhibitor promoter, lysozyme promoterand ovotransferrin promoter and functional portions of these promoters,e.g., promoter components.

The terms “polynucleotide,” “oligonucleotide,” “nucleotide sequence” and“nucleic acid sequence” can be used interchangeably herein and include,but are not limited to, coding sequences, i.e., polynucleotide(s) ornucleic acid sequence(s) which are transcribed and translated intopolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory or control sequences; controlling sequences,e.g., translational start and stop codons, promoter sequences, ribosomebinding sites, polyadenylation signals, transcription factor bindingsites, transcription termination sequences, upstream and downstreamregulatory domains, enhancers, silencers, DNA sequences to which atranscription factor(s) binds and alters the activity of a gene'spromoter either positively (induction) or negatively (repression) andthe like. No limitation as to length or to synthetic origin aresuggested by the terms described herein.

As used herein, the terms “polypeptide” and “protein” can be usedinterchangealy and refer to a polymer of amino acids of three or moreamino acids in a serial array, linked through peptide bonds. The term“polypeptide” includes proteins such as fusion proteins, proteinfragments, protein analogues, oligopeptides and the like. The term“polypeptides” includes polypeptides as defined above that are encodedby nucleic acids, produced through recombinant technology (e.g.,isolated from a transgenic bird), or chemically synthesized.

As used herein, the term “promoter” refers to a DNA sequence useful toinitiate transcription initiation by an RNA polymerase in an avian cell.A “promoter component” is a DNA sequence that can, by itself or, incombination with other DNA sequences effect or facilitate transcription.Specific promoter components such as ovalbumin promoter components,ovomucoid promoter components and lysozyme promoter components and otherpromoters and promoter components disclosed and claimed herein do notdescribe a specific promoter sequence. Rather, they encompass anysequence or sequence fragment of the respective promoter that is usefulto effect or facilitate transcription of a coding sequence. For example,an ovomucoid promoter component includes, without limitation, the about1.8 kb, the about 3.9 kb and the about 10 kb ovomucoid promotersdisclosed in U.S. Publication No. 11/649,543,published May 17 2007,whichis incorporated in its entirety herein by reference. “Promotercomponents” can also encompass rearranged gene expression controllingregions which function to initiate RNA transcription and hybrid DNAmolecules composed of naturally occurring DNA sequences and/or syntheticDNA sequences which function to initiate RNA transcription.

The terms “recombinant nucleic acid” and “recombinant DNA” as usedherein refer to combinations of at least two nucleic acid sequences thatare not naturally found in a eukaryotic or prokaryotic cell. The nucleicacid sequences may include, but are not limited to, nucleic acidvectors, gene expression regulatory elements, origins of replication,suitable gene sequences that when expressed confer antibioticresistance, protein-encoding sequences and the like. The term“recombinant polypeptide” or “recombinant protein” is meant to include apolypeptide produced by recombinant DNA techniques such that it isdistinct from a naturally occurring polypeptide either in its location,purity or structure. Generally, such a recombinant polypeptide will bepresent in a cell in an amount different from that normally observed innature.

The term “stringent conditions,” as used herein, is the “stringency”which occurs within a range from about Tm −5° C. (5° C. below themelting temperature (Tm) of the probe) to about 20° C. to 25° C. belowTm. As will be understood by those of skill in the art, the stringencyof hybridization may be altered in order to identify or detect identicalor related polynucleotide sequences.

As used herein, the term “subject” or “patient” encompasses mammals andnon-mammals. Examples of mammals include, but are not limited to,humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine;rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

As used herein, the term “therapeutically effective amount” refers toany amount of a compound which, as compared to a corresponding subjectwho has not received such amount, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of advancement of a disease ordisorder. The term also includes within its scope amounts effective toenhance normal physiological function.

As used herein, the terms “TAGNPP1,” “fusion protein,” “TAGNPP1polypeptide” and “NPP1 component fused to a targeting moiety” are usedinterchangeably.

As used herein, the term “treat,” “treating” or “treatment” refers tomethods of alleviating, abating or ameliorating a disease or conditionsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying causes of symptoms, inhibiting the disease or condition,arresting the development of the disease or condition, relieving thedisease or condition, causing regression of the disease or condition,relieving a condition caused by the disease or condition, or stoppingthe symptoms of the disease or condition either prophylactically and/ortherapeutically.

A “variant” of TAGNPP1,as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. Preferably, a variantcontains conservative substitutions. A “conservative substitution” isone in which an amino acid is substituted for another amino acid thathas similar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Amino acid substitutionsmay generally be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids include Aspand Glu; positively charged amino acids include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values include Leu, Ile and Val; Gly and Ala; Asp andGln; and Ser, Thr, Phe and Tyr. Other groups of amino acids that mayrepresent conservative changes include: Ala, Pro, Gly, Glu, Asp, Gln,Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe;(4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His. A variant may also, oralternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer, or by,for example, replacement of a Gly with a Trp. Variants may also (oralternatively) be modified by, for example, the deletion or addition ofamino acids that have minimal influence on the immunogenicity, secondarystructure and hydropathic nature of the NPP1 component. Guidance indetermining which amino acid residues can be substituted, inserted, ordeleted without abolishing biological or immunological activity can befound using computer programs well known in the art.

The term “vector” and “nucleic acid vector” as used herein refers to anatural or synthetic single or double stranded plasmid or viral nucleicacid molecule that can be transfected or transformed into cells andreplicate independently of, or within, the host cell genome. A circulardouble stranded vector can be linearized by treatment with anappropriate restriction enzyme based on the nucleotide sequence of thevector. A nucleic acid can be inserted into a vector by cutting thevector with restriction enzymes and ligating the desired piecestogether.

The term “portion,” as used herein, with regard to a fusion proteinrefers to fragments of that protein. The fragments may range in sizefrom four amino acid residues to the entire amino acid sequence minusone amino acid. Thus, a protein “comprising at least a portion of theamino acid sequence of SEQ ID NO:1” encompasses the full-length TAGNPP1and fragments thereof.

“Transformation” or “transfection,” as used herein, describes a processby which exogenous DNA enters and changes a recipient cell using variousmethods well known in the art. Transformation may rely on any knownmethod for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on thehost cell being transformed and may include, but is not limited to,electroporation, particle bombardment, viral infection, and lipofection.Such “transformed” cells include stably transformed cells in which theinserted DNA is capable of replicating either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

EXAMPLES

The present invention is further exemplified by the following examples.The examples are for illustrative purpose only and are not intended, norshould they be construed as limiting the invention in any manner.

Example I

The TAGsNNP1 construct containing the targeting moiety having eightconsecutive aspartic acids fused to sNPP1 was ligated into pTT22 vectorusing EcoRI and HindIII sites (pTT22-sNPP1.D8; FIG. 19). pTT22-sNPP1.D8was transfected into HEK203E cells and the transformants were culturedto express TAGsNNP1.TAGsNNP1 was isolated from the culture media andpartially purified as well known in the art. Following the purification,the pyrophosphase/phosphodiesterase activity of TAGsNPP1 was measuredfor its ability to hydrolyze thymmidine 5′ monophosphate p-nitrophenylester. Briefly, TAGsNPP1 was diluted to 1 ng/μL in 50 mM Tris, 250 mMNaCl, pH 9.5.In a plate containing 50 μL of 1 ng/μL TAGsNPP1, 50 μL of10 mM thymmidine 5′ monophosphate p-nitrophenyl ester (Sigma™, Catalog#T4510) substrate was added. The enzyme activity of TAGsNPP1 wasmeasured at 405 nm (absobance) in kinetic mode for 5 minutes. As shownin FIG. 21, the activity of TAGsNPP1 was detected above the levelobserved in control containing no TAGsNPP1. Particularly, TAGsNPP1produced from HEK203D6 exhibited the highest level of the enzymaticactivity. This results strongly suggested that the truncated NPP1 fusedto a targeting moiety (i.e., D8) sufficiently maintained its normalfunction as nuclease.

Example II

This non-limiting prophetic example describes how to treat idiopathicinfantile arterial calcification by administering a formulationcomprising a TAGNPP1 fusion protein.

A clinician uses a diagnostic test to verify that a patient has highlevels of calcification in the artery. A genetic test can be alsoperformed for NPP1 defects as described in Rutsch et al. (2003), NatureGenetics 34:379-81.

The pharmaceutical compositions of the present invention are preferablyadministered intravenously, although interadermal, intramuscular or oraladministration are employed in certain circumstances.

The clinician determines a dose which may vary depending on the gender,age, health, and weight of the patient. The determination of theappropriate dosage or route of administration is well within the skillof an ordinary physician.

The formulation containing TAGNPP1 can be infused, between about 10mg/kg and about 1000 mg/kg per week weekly. 10-30 mg/kg can beadministered once. During the infusion period, patients are monitoredclosely and appropriate clinical intervention is taken in the event ofan adverse event. Treatment lasts at least 1 month or for the life ofthe patient. A window of 48 hours may be allowed for each infusion. Aninfusion schedule in which the rate of infusion increases with timereduces or eliminates adverse events. Infusions for infants can beadministered according to the following schedule: 5-10 cc/hr for 60minutes in each interval.

On the other hand, when continuous intravenous administration isdesired, typical example of the slow release systems comprises that1-100 mg/kg of effective TAGNPP1 proteins can be continuously releasedfor more than 1 day.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated NPP1 fusion protein, comprising anNPP1 component, a targeting moiety, and an Fc region of animmunoglobulin, wherein a) the NPP1 component comprises thecysteine-rich region and the C-terminus catalytic domain of NPP1, b) thetargeting moiety and the Fc region are each located C-terminally to theNPP1 component, and c) the isolated NPP1 fusion protein has NPP1enzymatic activity.
 2. The NPP1 fusion protein of claim 1, wherein saidNPP1 enzymatic activity is pyrophosphatase activity, phosphodiesteraseactivity or pyrophosphatase and phosphodiesterase activity.
 3. The NPP1fusion protein of claim 1, wherein said polypeptide is a recombinantprotein.
 4. The NPP1 fusion protein of claim 1, wherein said targetingmoiety is chemically linked to said catalytic domain of NPP1.
 5. TheNPP1 fusion protein of claim 1, wherein said NPP1 component consists ofP99 through D925 of SEQ ID NO:
 1. 6. The NPP1 fusion protein of claim 5,wherein said targeting moiety is a peptide comprising at least fournegatively charged amino acid residues.
 7. The NPP1 fusion protein ofclaim 5, wherein said targeting moiety is a peptide comprising betweenabout five and about fifteen negatively charged amino acid residues. 8.The polypeptide NPP1 fusion protein of claim 7, wherein said negativelycharged amino acid residues comprise about eight consecutive asparticacid or glutamic acid residues.
 9. The NPP1 fusion protein of claim 7,wherein said negatively charged amino acid residues comprise at leastfour aspartic acid residues.
 10. The NPP1 fusion protein of claim 7,wherein said negatively charged amino acid residues comprise at leastfour glutamic acid residues.
 11. The NPP1 fusion protein of claim 7,wherein said negatively charged amino acid residues comprise eightconsecutive aspartic acid residues.
 12. The NPP1 fusion protein of claim5, wherein said targeting moiety is fused to the C-terminus of saidcatalytic domain of NPP1.
 13. The NPP1 fusion protein of claim 5,wherein said Fc region is fused to the C-terminus of said catalyticdomain of NPP1.
 14. The NPP1 fusion protein of claim 1, wherein saidfusion protein further comprises a polypeptide linker between saidtargeting moiety and said catalytic domain of NPP1.
 15. The NPP1 fusionprotein of claim 1, wherein said fusion protein further comprises asignal peptide.
 16. The NPP1 fusion protein of claim 1 herein saidfusion protein forms a homodimer.
 17. The NPP1 fusion protein of claim1, wherein said fusion protein protein is a monomer.
 18. An isolatednucleic acid encoding the NPP1 fusion protein of claim
 1. 19. Areplication or expression vector carrying the isolated nucleic acid ofclaim 18.